PROJECT SUMMARY The initiation stage of eukaryotic translation determines: (1) which mRNA is recruited to the ribosome for translation; and (2) which initiation codon is selected as the translation start site. Changes in physiological stimuli reprogram the translation machinery to alter preferential recruitment of mRNAs to the ribosome and which start site is selected. Genome wide analysis has shown that the regulation of these events is far more extensive than previously appreciated. Significant progress has been made in elucidating the mechanism of initiation codon selection, but the mechanism of mRNA recruitment to the ribosome and its regulation remains poorly understood. This knowledge gap persists because of difficulties in determining which intermediate step(s) in the initiation pathway function as kinetic checkpoints to control mRNA recruitment to the ribosome. To date, initiation pathway intermediates have been identified on the basis of their thermodynamic stability which must be enough to withstand traditional assays. These approaches take minutes to hours to perform, and/or require cross-linking agents to stabilize them, but most intermediates prior to initiation codon selection occur on the sub-second to second time scale. To determine how mRNAs are selected for translation, it will be necessary to generate new assays that can monitor the formation of pathway intermediates in real-time. To overcome this bottleneck, we have developed ensemble and single-molecule fluorescence-based assays that can observe the rate of mRNA recruitment to the human ribosome in real time. A highly purified complete reconstituted system that we have developed will form the cornerstone of this proposal. Models that we generate with this system will be tested using translation assays in cell-free extracts and intact cells. The long-term objective of this proposal is to understand the mechanism by which alterations in initiation factor availability and post- translational modification reprograms the translational apparatus to control which mRNAs are recruited to the ribosome in response to physiological stimuli. Aim 1. Characterize the mechanism by which human eIF4F binds to mRNA. Despite over three decades of research, the molecular details to explain how human eIF4F binds to the cap-proximal region of an mRNA is still poorly defined. The primary barrier that has prevented progress is the inability to generate recombinant full-length human eIF4G to carry out sophisticated kinetic assays. Our preliminary data has unexpectedly revealed that eIF4E possesses both cap-dependent and cap-independent functions in promoting mRNA binding to eIF4F. Aim 2. Identify and characterize kinetic checkpoints that control mRNA selection for translation. Early initiation pathway intermediates that control mRNA recruitment to the 40S subunit are poorly defined. It is possible that the accommodation of a mRNA into the mRNA entry channel of the 40S subunit commits it for translation. To rigorously test this, we will use fluorescent ensemble and single molecule assays to monitor real-time kinetic parameters of mRNA recruitment to the 40S subunit. Aim 3. Determine how mRNA secondary structure, initiation factor availability, and initiation factor phosphorylation control mRNA recruitment to the 40S subunit. The degree of secondary structure in a mRNA 5 UTR makes it more or less sensitive to changes in availability and phosphorylation of canonical initiation factors. Yet, the mechanism by which these changes reprogram the translational machinery to preferentially translate different mRNAs are not known.